|Year : 2013 | Volume
| Issue : 1 | Page : 51-56
c-Myc oncogene expression in selected odontogenic cysts and tumors: An immunohistochemical study
Zama Moosvi1, K Rekha2
1 Department of Oral Pathology, HKES' SN Dental College, Gulbarga, Karnataka, India
2 Department of Oral Pathology, Annoor Dental College, Ernakulam, Kerala, India
|Date of Web Publication||18-Apr-2013|
Central University of Karnataka, 2nd Floor - Karya Soudha, Gulbarga University Campus, Gulbarga - 585 106, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: To investigate the role of c-Myc oncogene in selected odontogenic cysts and tumors. Materials and Methods: Ten cases each of ameloblastoma, adenomatoid odontogenic tumor (AOT), odontogenic keratocyst (OKC), dentigerous cyst, and radicular cyst were selected and primary monoclonal mouse anti-human c-Myc antibody was used in a dilution of 1: 50. Statistical Analysis was performed using Mann Whitney U test. Results: 80% positivity was observed in ameloblastoma, AOT and OKC; 50% positivity in radicular cyst and 20% positivity in dentigerous cyst. Comparison of c-Myc expression between ameloblastoma and AOT did not reveal significant results. Similarly, no statistical significance was observed when results of OKC were compared with ameloblastoma and AOT. In contrast, significant differences were seen on comparison of dentigerous cyst with ameloblastoma and AOT and radicular cyst with AOT. Conclusion: From the above data we conclude that (1) Ameloblastoma and AOT have similar proliferative potential and their biologic behavior cannot possibly be attributed to it. (2) OKC has an intrinsic growth potential which is absent in other cysts and reinforces its classification as keratocystic odontogenic tumor.
Keywords: Adenomatoid odontogenic tumor, ameloblastoma, c-Myc oncogene, dentigerous cyst, immunohistochemistry, keratocystic odontogenic tumor, odontogenic keratocyst, radicular cyst
|How to cite this article:|
Moosvi Z, Rekha K. c-Myc oncogene expression in selected odontogenic cysts and tumors: An immunohistochemical study. J Oral Maxillofac Pathol 2013;17:51-6
|How to cite this URL:|
Moosvi Z, Rekha K. c-Myc oncogene expression in selected odontogenic cysts and tumors: An immunohistochemical study. J Oral Maxillofac Pathol [serial online] 2013 [cited 2021 Jun 14];17:51-6. Available from: https://www.jomfp.in/text.asp?2013/17/1/51/110725
| Introduction|| |
Odontogenic cysts and tumors, although originate from the same primitive odontogenic epithelium, differ widely in their pathogenesis and biologic behavior. Various concepts have been proposed which explain the pathogenesis of different odontogenic cysts and tumors. Studies using proliferative markers suggest that increased cell proliferation plays a role in the development of odontogenic cysts and tumors. This increased cell proliferation in odontogenic epithelium can result from perturbations in cell cycle regulators (Cyclin D1, p16 INK4a , p21 WAF/Cip1 , p27 Kip1 ), mutations in oncogenes (Ras, Myc, Fos), or tumor suppressor genes (p53, adenomatous polyposis coli (APC).  Genetic mutations have also been proposed as a possible cause in the development of odontogenic cysts like odontogenic keratocyst (OKC)  and dentigerous cyst.  Studies investigating the cell kinetics of epithelial linings of odontogenic cysts indicate that the lining of OKC may have some intrinsic growth potential which is not present in other cysts , and thus OKC is now regarded as a benign cystic neoplasm rather than a cyst. ,
The c-Myc protein is a nuclear transcription factor which centrally regulates cell proliferation, arrest, differentiation and death. By accelerating cells through G1 and S phases of the cell cycle, abrogating cell cycle checkpoints and increasing cell metabolism, c-Myc eventually renders genomic instability to the cells. ,,,, c-Myc also plays a role in the early stages of embryonic tooth development , and its expression here is related to the proliferation of odontogenic epithelial cells and its degree of differentiation.  Currently few data exist on the occurrence of c-Myc in odontogenic lesions. The aim of the present immunohistochemical study is to investigate the role of c-Myc oncoprotein in selected odontogenic cysts and tumors.
| Materials and Methods|| |
Fifty cases of buffered formalin fixed, routinely processed, paraffin embedded tissues, histologically diagnosed as odontogenic cysts and tumors were selected. These included ten cases of ameloblastoma, (five follicular and five plexiform), ten cases of adenomatoid odontogenic tumor (AOT), ten cases of OKC (only sporadic cases), ten cases of dentigerous cyst and ten cases of radicular cyst. Well differentiated squamous cell carcinoma was taken as positive control.
Sections of 4 μm thickness were taken on silanized slides (SIGMA diagnostics, USA), deparaffinized in xylene, hydrated through graded alcohol and washed in distilled water. Antigen retrieval was performed by heating slides immersed in 10 mM citrate buffer (pH 6.0) in a pressure cooker. Sections were brought to room temperature and the immunohistochemical procedure was performed. Endogenous peroxidase activity was blocked with 3% H 2 O 2 for 15 min. The sections were washed in tris buffered saline (TBS- wash solution) and incubated with primary monoclonal mouse anti-human c-Myc antibody (clone 9E10; Dako Cytomation, USA) at 4°C overnight, diluted to a ratio of 1:50 in TBS containing bovine serum albumin (TBS- antibody diluent solution) to block non-specific binding. The standard streptavidin-biotin-peroxidase complex method was performed to bind the primary antibody with the use of a Labelled streptavidin biotin (LSAB) + Visualization system - Horseradish peroxidase (HRP) (Dako Cytomation, USA) [Figure 1]. The slides were visualized by DAB solution and weakly counterstained with hematoxylin. Negative controls were prepared by replacing primary antibody with TBS and were confirmed to be unstained.
Evaluation and statistical analysis
Immunohistochemical expression of c-Myc oncoprotein was evaluated independently by three investigators. Presence of brown colored product at the site of target antigen was indicative of positive immunoreactivity for c-Myc expression. Staining reactions were evaluated and graded as positive or negative (0). The positive results were assessed further for intensity of staining as mild (1), moderate (2) and intense (3) staining and localization as either nuclear or cytoplasmic or nuclear and cytoplasmic staining. In cases with staining heterogeneity, the expression was grouped according to the predominant staining intensity.
The statistical significance of differences in the staining intensities between various study groups were analyzed by Mann Whitney U test and significance determined at 5% level of significance.
| Results|| |
The results of immunohistochemical staining of c-Myc in various study groups is summarized in [Table 1]. Among the odontogenic tumors studied, both follicular and plexiform ameloblastomas and adenomatoid odontogenic tumor expressed 80% positivity. In odontogenic cysts, OKC showed maximum number of cases with positive immunoreactivity (80%), followed by radicular cyst (50%) and dentigerous cyst (20%). [Table 2] shows mean and standard deviation values of intensity of c-Myc expression in the various study groups. Statistical comparison of staining intensity of c-Myc between different types of ameloblastoma and between ameloblastoma and AOT did not reveal any significance. Among the cysts, OKC showed staining intensity significantly higher than dentigerous cyst or radicular cyst while no significance was seen between dentigerous cyst and radicular cyst. When the staining results of the three cysts were compared individually with the tumors, the staining result of OKC was similar to that of ameloblastoma and AOT. Significant difference was observed in the staining intensities between dentigerous cyst and the tumours. Radicular cyst showed variable result with no significant difference when compared with ameloblastoma and a significant difference observed when compared with AOT [Table 3].
|Table 3: Comparison of intensity of c - Myc expression between different study groups using Mann Whitney U test |
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Reactivity for c-Myc protein in follicular and plexiform ameloblastomas was observed both in peripheral columnar ameloblast like cells and central polyhedral stellate reticulum like cells. Stellate reticulum like cells showed mainly nuclear staining while peripheral columnar ameloblast like cells showed both nuclear and cytoplasmic expression [Figure 2] and [Figure 3]. In AOT, predominantly nuclear staining was seen in whorls, ducts and sheets of epithelial cells [Figure 4]. Uniform staining was observed in all the layers of cystic lining in OKC, basal layer of cystic lining in dentigerous cyst and arcading epithelial lining in radicular cyst. Both nuclear and cytoplasmic staining was observed in the lining cells of all the cysts [Figure 5] and [Figure 6].
|Figure 2: Positive c-Myc expression showing intense staining in follicular ameloblastoma. Predominant nuclear staining of central polyhedral cells and cytoplasmic staining of peripheral columnar cells (×400).|
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|Figure 3: Positive c-Myc expression showing moderate staining in plexiform ameloblastoma (×250)|
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|Figure 4: Positive c-Myc expression showing moderate staining in adenomatoid odontogenic tumor (×250)|
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|Figure 5: Positive c-Myc expression showing intense staining in odontogenic keratocyst (×250)|
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| Discussion|| |
The etiology and pathogenesis of odontogenic tumors and cysts is a subject which is debated for decades. Various molecular alterations are now proposed to be responsible for the development of these lesions, one of which is the role of oncogenes. The role of c-Myc proto-oncogene in the regulation of normal cellular proliferation suggests that its deregulation contributes to deregulated DNA synthesis and genomic instability.  Overexpression of the c-Myc protein and its gene amplification has been identified in various neoplastic and non-neoplastic lesions. The fact that oncogene products can be detected by immunohistochemistry  and the advantage of maintaining the cellular and tissue architecture in this method  has prompted us to study the role of c-Myc oncogene product in selected odontogenic cysts and tumors.
The results of our study demonstrate a similar expression of c-Myc oncoprotein between ameloblastoma and AOT. Our findings are consistent with the argyrophilic nucleolar organizer regions counts studied in these two lesions. Since c-Myc expression is related to the proliferative status of the cells, our results in accordance with the findings of Carmo and Silva  suggest that the differences in the biologic behavior of ameloblastomas and AOT's are probably not related to their cellular proliferation ratio. In the ameloblastomas studied, there was no statistically significant difference observed in the expression of c-Myc between follicular and plexiform ameloblastomas which is in accordance with the findings of Kumamoto et al.  Reactivity for c-Myc protein in the follicular and plexiform ameloblastomas in our study was observed both in peripheral columnar cells and central polyhedral cells. The staining reaction of central polyhedral cells was mainly nuclear and that of peripheral columnar cells varied from being only nuclear to both nuclear and cytoplasmic. Our results are in compliance with those of Barawy et al.,  who also observed both nuclear and cytoplasmic staining. Considerable debate exists regarding the subcellular distribution of c-Myc oncogene product. Persson and Leder  have shown that c-Myc oncoprotein is predominantly found in the nucleus while Loke et al.,  have shown that though the c-Myc protein is predominantly localized in the nucleus, certain fixation procedures may allow these proteins to diffuse into the cytoplasm. They also observed that despite a variety of fixation methods, tissues either rich in hydrolytic enzymes or metabolically very active, exhibit cytoplasmic staining for c-Myc protein. The appearance of c-Myc protein in the nucleus and cytoplasm of the peripheral columnar ameloblast like cells in our study could be because these cells may be similar to the ameloblasts of the enamel organ which are metabolically very active whereas the predominant nuclear staining in the stellate reticulum like cells denotes its less activity when compared with the peripheral cells.
To date, data on the immunohistochemical expression of c-Myc oncoprotein in odontogenic cysts do not exist with only 1 study which determines the expression of c-Myc mRNA in OKC using in situ hybridization.  Our results showed 80% positivity for c-Myc expression in OKC which was significantly higher than dentigerous cyst (20%) and radicular cyst (50%). Previous studies assessing cell proliferation in the three major odontogenic cysts using various markers for proliferation like Proliferating cell nuclear antigen PCNA,  Ki67, , p53 , and Ipomeanol IPO-38,  have shown similar results wherein increased proliferative cells were observed in the lining of OKCs which was significantly higher than the dentigerous cyst and radicular cyst. While the expression of p53, Ki67, PCNA and IPO-38 was predominantly seen in the suprabasal layers, our results showed a uniform distribution of c-Myc expression in both basal and parabasal layers of the lining epithelium.
Comparison of c-Myc expression between OKC (80% +ve) and ameloblastoma (80% +ve) and OKC and AOT (80% +ve) in our study was not significant. Li et al.,  studied the expression of c-Myc mRNA and showed 75.0% positivity in OKC and 81.5% positivity in ameloblastoma. When OKC and ameloblastoma were compared for expression of PCNA,  IPO-38  and p53 , the percentage of positivity in the lining of OKC was not significantly different from those of ameloblastoma. Our findings are in accordance with other studies comparing epithelial cell proliferation between OKC and ameloblastoma. Collectively, these data explain that OKC has a proliferation index which is similar to those of ameloblastoma and AOT and significantly higher than in other kinds of odontogenic cysts. Hence its addition as 'keratocystic odontogenic tumor' seems to be justified.
Comparison of c-Myc expression between dentigerous cyst and ameloblastoma and dentigerous cyst and AOT in our study showed statistically significant differences. Previous comparative studies between dentigerous cyst and ameloblastoma by Thosaporn et al., on IPO-38,  Slootweg on p53  and Piattelli  using Ki67 have shown similar results. Together these data suggest that factors other than epithelial proliferation, like fluid accumulation in the follicle and contents of cystic fluid that increase the osmolality and hydrostatic pressure are of greater importance in the development and enlargement of dentigerous cyst.
Bando et al.,  showed that epithelial proliferation in the radicular cyst is stimulated by inflammatory cytokines (Interleukin-1) released by the inflammatory cells. Treatment with mitogens, including cytokines or growth factors such as interleukins results in a rapid increase in c-Myc mRNA and protein levels  and the expression of c-Myc in inflammatory gastric and colonic mucosa increased with greater degrees of inflammation.  Our results showed no statistically significant difference in the expression of c-Myc between ameloblastoma and radicular cyst, while a significant difference was observed when compared with AOT. This variable expression of c-Myc in radicular cyst could be attributed to inflammatory cytokines released by the inflammatory cells, which is a strong component in the development of radicular cysts.
| Conclusion|| |
The results of our study show a similar proliferative potential between ameloblastoma and AOT and thereby question the role of proliferation in determining the biologic behavior of these lesions. Among the cysts studied highest intensity of c-Myc expression was observed in OKC which was similar to the staining results of the tumors. This finding highlights the intrinsic growth potential present in the lining of OKC, and supports the hypothesis of OKC to be a benign cystic neoplasm rather than a cyst and attests for its addition under the benign odontogenic tumors as an entity termed 'keratinizing cystic odontogenic tumor'. However, further studies are needed using DNA assays to determine the role of c-Myc oncogene and any mutations if any which may play a role in the pathogenesis of these odontogenic lesions.
| Acknowledgment|| |
Dr. Kaveri H, Professor, Department of Oral Pathology, SDM College of Dental Sciences, Dharwad.
| References|| |
|1.||Kumamoto H. Molecular pathology of odontogenic tumors. J Oral Pathol Med 2006;35:65-74. |
|2.||Barreto DC, Gomez RS, Bale AE, Boson WL, De Marco L. PTCH gene mutations in odontogenic keratocysts. J Dent Res 2000;79:1418-22. |
|3.||Paveliæ B, Levanat S, Crniæ I, Kobler P, Aniæ I, Manojloviæ S, et al . PTCH gene altered in dentigerous cysts. J Oral Pathol Med 2001;30:569-76. |
|4.||Li TJ, Browne RM, Matthews JB. Quantification of PCNA+cells within odontogenic jaw cyst epithelium. J Oral Pathol Med 1994;23:184-9. |
|5.||Slootweg PJ. p53 protein and Ki-67 reactivity in epithelial odontogenic lesions. An immunohistochemical study. J Oral Pathol Med 1995;24:393-7. |
|6.||Reichart PA, Phillipsen HP. Odontogenic Tumours and Allied Lesions. London: Quintessence; 2004. |
|7.||Philipsen HP, Barnes L, Eveson JW, Reichart P, Sidransky D. WHO classification of Tumours. Pathology and Genetics of Head and Neck Tumours. Lyon: IARC Press; 2005. p. 306-7. |
|8.||Dang CV. c-Myc target genes involved in cell growth, apoptosis, and metabolism. Mol Cell Biol 1999;19:1-11. |
|9.||Persson H, Leder P. Nuclear localization and DNA binding properties of a protein expressed by human c-Myc oncogene. Science 1984;225:718-21. |
|10.||Studzinski GP, Brelvi ZS, Feldman SC, Watt RA. Participation of c-Myc protein in DNA synthesis of human cells. Science 1986;234:467-70. |
|11.||Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Brooks M, et al. Induction of apoptosis in fibroblasts by c-Myc protein. Cell 1992;69:119-28. |
|12.||Gardner L, Lee L, Dang C. The c-Myc oncogenic transcription factor (Adapted from myc oncogene). Encyclopedia of Cancer. 2 nd ed.: Academic Press; 2002. |
|13.||Pfeifer-Ohlsson S, Rydnert J, Goustin AS, Larsson E, Betsholtz C, Ohlsson R. Cell-type-specific pattern of myc protooncogene expression in developing human embryos. Proc Natl Acad Sci U S A 1985;82:5050-4. |
|14.||Schmid P, Schulz WA, Hameister H. Dynamic expression pattern of the myc protooncogene in midgestation mouse embryos. Science 1989;243:226-9. |
|15.||Barawy SE, Raslan HS, Riad SE. Expression of c-Myc oncoprotein in benign and malignant ameloblastomas official. J Egypt Dent Assoc 2001;47:1765-9. |
|16.||Ciclitira PJ, Macartney JC, Evan G. Expression of c-Myc in non-malignant and pre-malignant gastrointestinal disorders. J Pathol 1987;151:293-6. |
|17.||do Carmo MA, Silva EC. Argyrophilic nucleolar organizer regions (AgNORs) in ameloblastomas and adenomatoid odontogenic tumours (AOTs). J Oral Pathol Med 1998;27:153-6. |
|18.||Kumamoto H, Kinouchi Y, Ooya K. Telomerase activity and telomerase reverse transciptase (TERT) expression in ameloblastomas. J Oral Pathol Med 2001;30:231-6. |
|19.||Loke SL, Neckers LM, Schwab G, Jaffe ES. c-Myc protein in normal tissue. Effects of fixation on its apparent subcellular distribution. Am J Pathol 1988;131:29-37. |
|20.||Li ZJ, Zhong M, Wang J, Zhang B, Hou L. Research on the expression of c-Myc mRNA in ameloblastoma. Shanghai J Stomatol 2004;13:515-8. |
|21.||Li TJ, Browne RM, Matthews JB. Epithelial cell proliferation in odontogenic keratocysts: A comparative immunocytochemical study of Ki67 in simple, recurrent and basal cell naevus syndrome (BCNS)-associated lesions. J Oral Pathol Med 1995;24:221-6. |
|22.||Piattelli A, Fioroni M, Santinelli A, Rubini C. P53 protein expression in odontogenic cysts. J Endod 2001;27:459-61. |
|23.||Thosaporn W, Iamaroon A, Pongsiriwet S, Ng KH. A comparative study of epithelial cell proliferation between the odontogenic keratocyst, orthokeratinized odontogenic cyst, dentigerous cyst, and ameloblastoma. Oral Dis 2004;10:22-6. |
|24.||Takahashi H, Fujita S, Yamabe S, Moriishi T, Okabe H, Tajima Y, et al. Comparison of proliferating cell nuclear antigen expression in odontogenic keratocyst and ameloblastoma: An immunohistochemical study. Anal Cell Pathol 1998;16:185-92. |
|25.||Piattelli A, Lezzi G, Fioroni M, Santinelli A, Rubini C. Ki-67 expression in dentigerous cysts, unicystic ameloblastomas, and ameloblastomas arising from dental cysts. J Endod 2002;28:55-8. |
|26.||Bando Y, Henderson B, Meghji S, Poole S, Harris M. Immunocytochemical localization of inflammatory cytokines and vascular adhesion receptors in radicular cysts. J Oral Pathol Med 1993;22:221-7. |
|27.||Ayer DE, Eisenman RN. C-Myc protooncogene. Encyclopedia of Cancer, Vol 1. San Diego: Academic Press; 1997. p. 419-31. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]